A novel direct adenosine monophosphate kinase activator ameliorates disease progression in preclinical models of Autosomal Dominant Polycystic Kidney Disease
Authors: Pascale Gluais Dagorn, Bjoern Buchholz, Andre Kraus, Battsetseg Batchuluun, Hester Bange, Laura Blockken, Gregory R. Steinberg, David E. Moller, Sophie Hallakou-Bozec
Journal: Kidney International (2023)

Autosomal dominant polycystic kidney disease (ADPKD) mainly results from mutations in the PKD1 gene, which encodes polycystin 1. It is the most common inherited kidney disease and is characterized by a progressive bilateral increase in cyst number and size, often leading to kidney failure. The cellular energy sensor and regulator adenosine monophosphate stimulated protein kinase (AMPK) has been implicated as a promising new therapeutic target. To address this hypothesis, we determined the effects of a potent and selective clinical stage direct allosteric AMPK activator, PXL770, in canine and patient-derived 3D cyst models and an orthologous mouse model of ADPKD. PXL770 induced AMPK activation and dose-dependently reduced cyst growth in principal-like Madin-Darby Canine Kidney cells stimulated with forskolin and kidney epithelial cells derived from patients with ADPKD stimulated with desmopressin. In an inducible, kidney epithelium-specific Pkd1 knockout mouse model, PXL770 produced kidney AMPK pathway engagement, prevented the onset of kidney failure (reducing blood urea by 47%), decreased cystic index by 26% and lowered the kidney weight to body weight ratio by 35% compared to untreated control Pkd1 knockout mice. These effects were accompanied by a reduction of markers of cell proliferation (-48%), macrophage infiltration (-53%) and tissue fibrosis (-37%). Thus, our results show the potential of direct allosteric AMPK activation in the treatment of ADPKD and support the further development of PXL770 for this indication. Autosomal dominant polycystic kidney disease (ADPKD) mainly results from mutations in the PKD1 gene, which encodes polycystin 1. It is the most common inherited kidney disease and is characterized by a progressive bilateral increase in cyst number and size, often leading to kidney failure. The cellular energy sensor and regulator adenosine monophosphate stimulated protein kinase (AMPK) has been implicated as a promising new therapeutic target. To address this hypothesis, we determined the effects of a potent and selective clinical stage direct allosteric AMPK activator, PXL770, in canine and patient-derived 3D cyst models and an orthologous mouse model of ADPKD. PXL770 induced AMPK activation and dose-dependently reduced cyst growth in principal-like Madin-Darby Canine Kidney cells stimulated with forskolin and kidney epithelial cells derived from patients with ADPKD stimulated with desmopressin. In an inducible, kidney epithelium-specific Pkd1 knockout mouse model, PXL770 produced kidney AMPK pathway engagement, prevented the onset of kidney failure (reducing blood urea by 47%), decreased cystic index by 26% and lowered the kidney weight to body weight ratio by 35% compared to untreated control Pkd1 knockout mice. These effects were accompanied by a reduction of markers of cell proliferation (-48%), macrophage infiltration (-53%) and tissue fibrosis (-37%). Thus, our results show the potential of direct allosteric AMPK activation in the treatment of ADPKD and support the further development of PXL770 for this indication. Translational StatementAdenosine monophosphate activated protein kinase (AMPK) has emerged as a target of interest for the treatment of autosomal dominant polycystic kidney disease (ADPKD). PXL770 is a clinical stage direct and specific allosteric AMPK activator; it has shown translation of several effects of AMPK activation from rodent models to humans (with nonalcoholic fatty liver disease and with diabetes). Here, PXL770 demonstrated efficacy in vitro in human cysts obtained from a patient with ADPKD as well as in vivo in a well-validated ADPKD mouse model that has shown translation to humans for tolvaptan, the only approved drug for ADPKD. These results suggest that PXL770 is a promising candidate for the treatment of ADPKD. Adenosine monophosphate activated protein kinase (AMPK) has emerged as a target of interest for the treatment of autosomal dominant polycystic kidney disease (ADPKD). PXL770 is a clinical stage direct and specific allosteric AMPK activator; it has shown translation of several effects of AMPK activation from rodent models to humans (with nonalcoholic fatty liver disease and with diabetes). Here, PXL770 demonstrated efficacy in vitro in human cysts obtained from a patient with ADPKD as well as in vivo in a well-validated ADPKD mouse model that has shown translation to humans for tolvaptan, the only approved drug for ADPKD. These results suggest that PXL770 is a promising candidate for the treatment of ADPKD. Autosomal dominant polycystic kidney disease (ADPKD) is the fourth leading cause of kidney failure and the most common inherited kidney disease.1Bergmann C. Guay-Woodford L.M. 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However, these approaches may not qualify for long-term clinical use because of anticipated adverse effects, inadequate potency, and a lack of specificity. To date, the effects of any small molecules that directly and specifically bind to and activate AMPK have not yet been tested. PXL770 is a potent, direct, and highly selective small molecule, activating AMPK allosterically by binding to the allosteric drug and metabolite site. In addition, it prevents AMPK from dephosphorylation by protein phosphatase 2C.33Gluais-Dagorn P. Foretz M. Steinberg G.R. et al.Direct AMPK activation corrects NASH in rodents through metabolic effects and direct action on inflammation and fibrogenesis.Hepatol Commun. 2022; 6: 101-119Crossref PubMed Scopus (26) Google Scholar PXL770 shows a substantially higher potency for AMPK complexes that contain the β1 subunit; this is important in a context of kidney disease, as the AMPK α2β1γ1 complex is predominantly expressed in the kidney of both humans and rodents.14Song X. Tsakiridis E. Steinberg G.R. Pei Y. Targeting AMP-activated protein kinase (AMPK) for treatment of autosomal dominant polycystic kidney disease.Cell Signal. 2020; 73109704Crossref Scopus (17) Google Scholar PXL770 is the first direct AMPK activator to have progressed to clinical efficacy studies. In 2 trials involving subjects with nonalcoholic fatty liver disease, we have recently shown the evidence of adequate safety, suitable pharmacokinetics, and systemic target engagement.34Cusi K. Alkhouri N. Harrison S.A. et al.Efficacy and safety of PXL770, a direct AMP kinase activator, for the treatment of non-alcoholic fatty liver disease (STAMP-NAFLD): a randomised, double-blind, placebo-controlled, phase 2a study.Lancet Gastroenterol Hepatol. 2021; 6: 889-902Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,35Fouqueray P. Bolze S. Dubourg J. et al.Pharmacodynamic effects of direct AMP kinase activation in humans with insulin resistance and non-alcoholic fatty liver disease: a phase 1b study.Cell Rep Med. 2021; 2100474PubMed Google Scholar Here, we assessed the effects of PXL770 on in vitro cyst growth using established cell-based models and we studied in vivo effects in a tamoxifen-induced Pkd1 knockout mouse model. The results of these studies yielded important evidence of efficacy, collectively establishing preclinical validation for direct AMPK activation in ADPKD, suggesting that PXL770 is a promising clinical candidate for the treatment of this severe genetic form of chronic kidney disease. Principal-like Madin-Darby Canine Kidney cells36Buchholz B. Teschemacher B. Schley G. et al.Formation of cysts by principal-like MDCK cells depends on the synergy of cAMP- and ATP-mediated fluid secretion.J Mol Med (Berl). 2011; 89: 251-261Crossref PubMed Scopus (42) Google Scholar were cultured at 37 °C, 21% O2, and 5% CO2 in a modified Eagle minimal essential medium, containing “Earle’s balanced salt solution,” 2 mM l-glutamine, 10% heat shock–inactivated fetal calf serum, 50 IU/ml of penicillin, and 50 μg/ml of streptomycin. Cell viability was assessed as described in Supplementary Methods. For cyst formation, principal-like Madin-Darby Canine Kidney cells were trypsinized and resuspended as a single-cell suspension of 104 cells/ml in Type I collagen solution (PureCol, CellSystems GmbH) before being transferred to 24-well plates. Next, cell culture medium was added, containing either 10 μM forskolin (Sigma) or 10 μM forskolin plus PXL770 (Poxel SA). PXL770 and metformin (Sigma) were dissolved in dimethylsulfoxide. The experiments were performed over a 5-day period of exposure to drug or control medium. Thereafter, photos were taken in 4 different areas of each well (at 0, 3, 6, and 9 o’clock) using the Zeiss Primovert microscope with the Zeiss Axiocam 105 color camera (both Zeiss Microscopy). Then, cyst diameters were measured with ImageJ software (version 1.48; https://imagej.net/ij/index.html). By assuming a spherical shape of the cysts, volumes were calculated by the use of the formula (4/3)πr3. The investigators were blinded to the experimental conditions. Human kidney cells were obtained via nephrectomy from a patient with ADPKD (Pkd1 mutation: c.5622G>A p.Trp1874∗; kindly provided by the Leiden University Medical Center). Cells were mixed with PrimCyst-Gel (Crown Bioscience Netherlands BV). The cell-gel mixture was then pipetted to 384-well plates to a final cell density of 450 cell clusters per well. After gel polymerization, cell culture medium was added. Cells were grown in the gel matrix for 24 hours, after which newly formed cysts were exposed for 48 hours to vehicle (0.2% dimethylsulfoxide plus phosphate-buffered saline), 2.5 μM desmopressin (ddAVP [1-desamino-8-d-arginine-vasopressin], Tocris) to induce cyst swelling, 2.5 μM desmopressin plus PXL770 or tolvaptan (Merck) or metformin (Selleckchem) at given concentrations, or 0.25 μM staurosporine (Selleckchem). Cysts were fixed with 4% formaldehyde (Sigma Aldrich), permeabilized with 0.2% Triton X-100 (Sigma-Aldrich), and stained with 0.25 μM rhodamine-phalloidin (F-actin staining, Sigma-Aldrich) and 0.1% Hoechst 33258 (nuclei staining, Sigma-Aldrich) for 2 days. Imaging was done using ImageXpress Micro XLS (Molecular Devices). For each well, ∼35 images in the Z direction were made for both channels, capturing the whole z plane for the whole well in each image. Image analysis was performed using Ominer software (Crown Bioscience Netherlands BV). The automated calculation of cyst area and cytotoxicity was performed as previously described37Booij T.H. Bange H. Leonhard W.N. et al.High-throughput phenotypic screening of kinase inhibitors to identify drug targets for polycystic kidney disease.SLAS Discov. 2017; 22: 974-984Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar (details in Supplementary Methods). Principal-like Madin-Darby Canine Kidney cells were cultured as described above with 10 μM forskolin until reaching a confluency of 70% to 80%, followed by a 24-hour treatment with 50 μM PXL770. Human cysts were cultured as described above in 6-well plates to a final cell density of 1 million cells per well; 25 μM of PXL770 was added in the presence of 2.5 μM desmopressin. Proteins were extracted for Western blot experiments. Proteins were transferred on a membrane (Millipore) and probed with primary antibodies against phosphorylated AMPK (p-AMPK; #2535; Cell Signaling), AMPKα (#2532; Cell Signaling), phosphorylated acetyl-CoA carboxylase (p-ACC; #3661; Cell Signaling), and ACC (#3676; Cell Signaling). Standard operating procedures and methods described in the AAALAC accreditation guide and approved by the animal experimentation committee were used. The tamoxifen-inducible, kidney epithelium–specific Pkd1 deletion mouse model carrying the loxP-flanked conditional alleles of Pkd1 (KspCad-CreERT2, Pkd1lox,lox) was described previously.38Lantinga-van Leeuwen I.S. Leonhard W.N. van der Wal A. et al.Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice.Hum Mol Genet. 2007; 16: 3188-3196Crossref PubMed Scopus (164) Google Scholar Twenty-two male mice per group were treated with tamoxifen (150 mg/kg by oral gavage) on postnatal days 18, 19, and 20 (“Pkd1 conditional knockout [cKO] mice”). PXL770 treatment (Poxel SA), 75 mg/kg twice daily or vehicle (0.5 w/v% carboxymethylcellulose with Tween 80 [98/2]), was administered by oral gavage starting at postnatal day 42. One group of 10 mice (designated as wild type [WT]) did not receive tamoxifen and was treated only with vehicle. Blood urea was measured weekly starting at postnatal day 75 by using the urea Reflotron Plus reagent strip (Roche). All mice with blood urea levels >20 mmol/l were considered at high risk of developing end-stage renal disease and were killed for ethical reasons when meeting this criterion. When ∼50% of untreated Pkd1 cKO mice reached this threshold, all remaining mice were killed. Kidneys were removed, weighed for the calculation of 2-kidney to body weight ratio, and snap frozen in liquid nitrogen or fixed in 4% formaldehyde. Left kidneys were sectioned in the transverse direction and stained using hematoxylin and eosin. The slides were scanned digitally using the SLIDEVIEW VS200 scanner (Olympus). The region of interest comprising the whole kidney cortex was defined using ImageJ software (version 1.53) and the Cintiq 13HD creative pen display (Wacom). Normal tubule space and cystic area were separated by defining diameters of noncystic tubules <50 μm using ImageJ software. The whole cortex cyst area was then divided by the whole cortex area and defined as the cystic index. Immunohistochemistry experiments were performed to quantitate cell proliferation (Ki67 antibody), macrophage infiltration (F4/80 antibody), and fibrosis (Sirius red coloration). The relative abundance of collecting duct versus proximal tubule cells was also assessed using Dolichos biflorus agglutinin (DBA) and Lotus-tetragonolobus lectin, respectively. Details are provided in Supplementary Methods. Homogenates from 11 to 12 kidneys per group were prepared using 1X lysis buffer containing phosphatase inhibitors (S6508, Sigma) for Western blot experiments. Proteins were separated using sodium dodecylsulfate–polyacrylamide gel electrophoresis (7.5%) Bis/Tris, 3-(N-morpholino) propanesulfonic acid running buffer. Separated proteins were transferred to polyvinylidene diflouride membranes. Nonspecific binding was blocked, and membranes were probed with antibodies against phospho-T172AMPK (#2535; Cell Signaling), AMPKα (#2532; Cell Signaling), phospho-ribosomal protein S6 kinase (p70S6K; #9234, Thr389 antibody; Cell Signaling Technology), and p70S6K (#9202; Cell Signaling Technology) or β-actin (#5125S, Cell Signaling Technology). Total DNA was extracted from kidneys using the SpeeDNA Isolation Kit (ScienCell Research Laboratories). The absolute copy number of mitochondrial DNA was quantified using the qPCR Assay Kit (ScienCell Research Laboratories). Total RNA was extracted using the RNeasy Kit (74106, Qiagen), and PGC-1α mRNA levels were assessed using a quantitative polymerase chain reaction (details in Supplementary Methods). All results are expressed as mean ± SEM. Statistical analysis was performed with GraphPad Prism 9.2.0. Comparisons between groups were done using 1-way analysis of variance followed by the Tukey or Dunnett multiple comparison test when normal distribution could be assumed; otherwise, the nonparametric Kruskal-Wallis test was used. Comparisons between 2 groups used the Student t test. Results were considered statistically significant when P < 0.05. pIMDCK cells resemble principal cells of the collecting duct, form cysts within a collagen 1 matrix, and enlarge in the presence of forskolin, an inducer of cyclic adenosine monophosphate production.36Buchholz B. Teschemacher B. Schley G. et al.Formation of cysts by principal-like MDCK cells depends on the synergy of cAMP- and ATP-mediated fluid secretion.J Mol Med (Berl). 2011; 89: 251-261Crossref PubMed Scopus (42) Google Scholar In this model, PXL770 activated AMPK as indicated by increased phosphorylation of AMPK at αT172 and ACC at Ser79 (Figure 1a). PXL770 dose dependently and significantly inhibited pIMDCK cyst growth compared to untreated cells (-30%, -65%, and -82% at 10, 25, and 50 μM, respectively; Figure 1b and c) with no change in the number of dead cells (Supplementary Figure S1A). In contrast, 10 μM metformin had no effect on cyst growth while higher concentrations (100 μM and 1 mM) elicited only modest inhibitory effects on cyst growth compared with PXL770 (Figure 1c). The potential of PXL770 to affect human cyst growth was assessed using post-nephrectomy kidney cells obtained from a patient with ADPKD. These cells form cysts when cultured in a 3-dimensional environment with stimulation by addition of desmopressin (ddAVP) promoting cyst swelling.39Kanhai A.A. Bange H. Verburg L. et al.Renal cyst growth is attenuated by a combination treatment of tolvaptan and pioglitazone, while pioglitazone treatment alone is not effective.Sci Rep. 2020; 10: 1672Crossref PubMed Scopus (18) Google Scholar PXL770 significantly inhibited cyst growth at 15 and 25 μM (∼-40% vs. ddAVP control; Figure 2a and b), with complete cyst area normalization at 50 μM (Figure 2a and b). The calculated 50% inhibitory concentration (IC50) was 12 μM. Similar results were achieved with tolvaptan, although only the highest concentration of 5 μM was statistically significant. In contrast, metformin had no effect on cyst growth (Supplementary Figure S2). No cell death with PXL770 or tolvaptan was observed (Supplementary Figure S1B). The direct effect of PXL770 on cell proliferation was also assessed in this system by counting the number of nuclei per cyst to evaluate the cell number (Supplementary Figure S3A). As expected, ddAVP significantly induced cell proliferation as measured by the increase in the number of cells by 32% compared with control dimethylsulfoxide. PXL770 at 50 μM produced a significant effect to prevent proliferation induced by ddAVP. Tolvaptan also prevented cell proliferation induced by ddAVP at the highest concentration. Similar to canine cells, PXL770 (25 μM) significantly increased the phosphorylation of ACC by 3.2-fold compared with vehicle control (Figure 2c) while there was a trend toward increased phosphorylation of AMPK at αT172 (Supplementary Figure S3B). These data are consistent with the effects of PXL770 being an allosteric activator of AMPK where increases in αT172 phosphorylation are not required for activation33Gluais-Dagorn P. Foretz M. Steinberg G.R. et al.Direct AMPK activation corrects NASH in rodents through metabolic effects and direct action on inflammation and fibrogenesis.Hepatol Commun. 2022; 6: 101-119Crossref PubMed Scopus (26) Google Scholar (reviewed in Steinberg an